This relates to human antigens that can be used for the diagnosis of myositis and myositis-overlap syndromes that have an autoimmune pathogenesis and more particularly relates to the Mi-2 and PM-Scl antigens.
Autoimmune disorders arise when the immune system reacts against its own tissues. Autoimmune diseases are often classified on the basis of whether a single organ or tissue is involved or whether multiple organs or tissues are involved. Generalized or systemic autoimmune diseases, such as systemic lupus erythematosus (SLE), characterized by the involvement of multiple organs and tissues, are often associated with the presence of autoantibodies to fundamental cellular components. Other autoimmune diseases are characterized by autoantibodies to antigens associated with a single organ or tissue.
Systemic autoimmune diseases are typically characterized by the presence of autoantibodies. Some of the autoantibodies associated with the particular disease may be disease specific and others may be common to many autoimmune diseases. For example, SLE, which is a prototypical immune disorder, is characterized by the presence of autoantibodies that are detectable in other autoimmune disease, such as anti-single-strand DNA antibodies, anti-histones antibodies, and anti-ribonuclear particle (RNP) antibodies, and also by the presence of autoantibodies that are SLE-specific, such as the anti-double-stranded DNA antibodies. Other systemic autoimmune disorders, such as rheumatoid arthritis and idiopathic inflammatory myopathies, are also characterized by the presence of autoantibodies in the sera of patients that react with fundamental nuclear and cytoplasmic intracellular components. As with SLE, some of these autoantibodies are associated with other autoimmune disorders and some are specifically associated with autoimmune myositis.
The idiopathic inflammatory myopathies polymyositis, dermatomyositis and the related overlap syndromes disorder, such as polymyositis-scleroderma overlap, are inflammatory myopathies that are characterized by chronic muscle inflammation and proximal muscle weakness. The muscle inflammation causes muscle tenderness, muscle weakness, and ultimately muscle atrophy and fibrosis as described by Plotz et al., Annals of Internal Med. 111:143-157 (1989). Also associated with the muscle inflammation are elevated serum levels of aldolase, creatine kinase, transaminases (such as alanine aminotransferase and aspartate aminotransferase) and lactic dehydrogenase. Other systems besides muscle can be affected by these conditions, resulting in arthritis, Raynaud""s phenomenon, and interstitial lung disease. Clinically, polymyositis and dermatomyositis are distinguished by the presence of a characteristic rash in patients with dermatomyositis. Differences in the myositis of these conditions can be distinguished in some studies of muscle pathology.
Autoantibodies can be detected in about 90% of patients with polymyositis and dermatomyositis according to Reichlin and Arnett, Arthritis and Rheum. 27:1150-1156 (1984). Sera from about 60% of these patients form precipitates with bovine thymus or human spleen extracts on Ouchterlony immunodiffusion (ID), while sera from about 80% of these patients stain tissue culture substrates, such as HEp-2 cells, by indirect immunofluorescence (IIF) (Targoff and Reichlin, Arthritis and Rheum. 28:796-803 (1985); Nishikai and Reichlin Arthritis and Rheum. 23:881-888 (1980); Reichlin et al., J. Clin. Immunol. 4:40-44 [1984]). There are numerous precipitating autoantibody specificities in myositis patients, but each individual antibody specificity occurs in only a fraction of the patients.
Many autoantibodies associated with myositis or myositis-overlap syndrome have been defined and in some cases the antibodies have been identified. These include antibodies that are present in other disorders and also disease-specific antibodies as described by Targoff and Reichlin, Mt. Sinai J. of Med. 55:487-493 (1988). Characteristic antibodies and their respective specificities are listed in Table 1. For example, a group of myositis-associated autoantibodies have been identified which are directed at cytoplasmic proteins that are related to tRNA and protein synthesis, particularly aminoacyl-tRNA synthetases. These include anti-Jo-1, which is directed against histidyl-tRNA synthetase and is the most common autoantibody associated with myositis autoimmune disorders (about 20% of such patients according to Nishikai and Reichlin, Arthritis Rheum. 23:881-888 [1980]); anti-PL-7, which is directed against threonyl-tRNA synthetase; and anti-PL-12, which is directed against alanyl-tRNA synthetase. A characteristic group of features is associated with anti-synthetases (Love et al., Medicine 70:360-374 [1991]). Anti-U1 RNP, which is frequently found in patients with SLE, may also be found in mixed connective tissue disease, overlap syndromes involving myositis, or in some cases of myositis alone. This antibody reacts with proteins that are uniquely present on the U1 small nuclear ribonucleoprotein, one of the nuclear RNPs that are involved in splicing mRNA. Autoantibodies that are associated with other conditions are sometimes found in patients with overlap syndrome such as anti-Sm, anti-Ro/SSA and anti-La/SSB. Anti-Ku has been found in myositis-scleroderma overlap syndrome and in SLE. The Ku antigen is a DNA binding protein complex with two polypeptide components, both of which have been cloned. Anti-Jo-1 and other anti-synthetases are disease-specific. Other myositis-associated antibodies are anti-PM-Scl, which is present in about 5-10% of myositis patients, many of whom have polymyositis-scleroderma overlap, and anti-Mi-2, which is present in about 8% of myositis patients, almost exclusively in dermatomyositis. Anti-Mi-2 is found in high titer in about 20% of all dermatomyositis patients and in low titer, by ELISA only, in less than 5% of polymyositis patients (Targoff and Reichlin, Mt. Sinai J. of Med. 55:487-493 [1988]).
Anti-Mi was first described by Reichlin and Mattioli, Clin. Immunol. and Immunopathol. 5:12-20 (1976). A complement-fixation reaction was used to detect it and, in that study, patients with dermatomyositis, polymyositis and polymyositis overlap syndrome had positive reactions. The prototype or reference serum, from patient Mi, forms two precipitin lines on immunodiffusion (ID) with calf thymus antigens, Mi-1 and Mi-2. Mi-1, which has been purified from bovine thymus nuclear extracts (Nishikai et al., Mol. Immunol. 17:1129-141 [1980]) is rarely found in other sera and is not myositis specific (Targoff et al., Clin. Exp. Immunol. 53:76-82 [1983]).
Anti-Mi-2 was found to be a myositis-specific autoantibody by Targoff et al., Arthritis and Rheum. 28:796-803 (1985). Furthermore, all patients with the precipitating antibody have the dermatomyositis rash. It is therefore potentially important as a diagnostic tool and, perhaps, ultimately as a tool for understanding the disease etiology. Anti-Mi-2 is also the only antibody response that appears to be selective for dermatomyositis and not for other subgroups of inflammatory myopathy without skin involvement.
Bovine thymus Mi-2 antigen was originally found to be a nuclear protein that separates in SDS polyacrylamide (SDS-PAGE) gels into two bands with apparent molecular weights of 53 kilodaltons (hereinafter kDa) and 61 KDa, respectively. Recently, additional higher molecular weight bands have been found. The bovine thymus antigenic activity is destroyed by SDS-PAGE and is trypsin sensitive, but not RNAse sensitive (Targoff et al., Arthritis and Rheum. 28:796-803 [1985]). Its nature and function have not as yet been identified.
Anti-PM-1 was first identified as an antibody found in 61% of dermatomyositis/polymyositis patients, including patients with polymyositis-scleroderma overlap (Wolfe et al., J. Clin. Invest. 59:176-178 [1977]). Anti-PM-1 was subsequently shown to be more than one antibody. The unique specificity component of anti-PM-1 was later named anti-PM-Scl (Reichlin et al., J. Clin. Immunol. 4:40-44 [1984]). Anti-PM-Scl is found in the sera of about 5-10% of myositis patients, but is most commonly associated with polymyositis-scleroderma overlap syndrome. It also occurs in patients with polymyositis or dermatomyositis alone or in patients with scleroderma without myositis.
Anti-PM-Scl antibody immunoprecipitates a complex from HeLa cell extracts of at least eleven polypeptides that have molecular weights ranging from about 20 to 110 kDa as described by Reimer et al., J. Immunol. 137:3802-3808 (1986), and possibly up to 16 polypeptides as described by Gelpi et al., Clin. Exp. Immunol. 81:59-64 (1990). The antigen is trypsin-sensitive, occurs in nucleoli (Targoff and Reichlin, Arthritis Rheum. 28:226-230 [1985]) and is believed to be a pre-ribosomal particle.
In an abstract, Bluthner, et al., First Int. Workshop on the Mol. and Cell Biology of Autoantibodies and Autoimmunity in Heidleberg (Springer-Verlag, Jul. 27-29, 1989) reported that sera from patients suffering from polymyositis/scleroderma-overlap syndrome (PM/Scl) recognize two major nucleolar proteins of 95 and 75 kDa molecular weight in Western blots of a HeLa cell extract. They also reported that cDNA that encodes a 20 kDa protein reactive with autoantibodies eluting from the 95 kDa PM-Scl HeLa antigen subunit had been cloned from a HeLa cDNA library.
Alderuccio et al., J. Exp. Med. 173:941-952 (1991), have cloned and sequenced the 75 kDa component of the PM-Scl antigen. The xe2x80x9c75 kDaxe2x80x9d was found to be a protein of 39.2 kDa that migrates aberrantly on polyacrylamide gel electrophoresis (PAGE) because of a region that is rich in acidic residues at the carboxyl half of the molecule.
The antibodies set forth in Group A of Table 1 serve as useful diagnostic markers because of their high specificity for myositis and its subgroups. At the present time, however, it is difficult and time consuming to routinely screen sera for the presence of these antibodies because standard serum needed for comparison is not widely available and highly concentrated tissue extracts must be used and the technique of immunodiffusion is slow and insensitive. Both anti-PM-Scl and anti-Mi-2 give only weak reactions in immunodiffusion, making them even more difficult to detect. In addition, these screening assays generally use the corresponding bovine antigen (which is more readily available for clinical purposes), which may not detect the presence of autoantibodies that do not cross-react sufficiently to be detectable.
There is, thus, a need to obtain the human myositis-specific antigens, such as the Mi-2 antigen and the PM-Scl antigen, in purified form, and plentiful, readily available amounts so that rapid, accurate, and convenient diagnostic assays can be developed. Recent studies with other antigens such as anti-Jo-1 have indicated a correlation of antibody level with disease activity, as reported by Miller, et al., J. Clin. Invest. 85:468-475 (1990). Quantitative assays for these antibodies may help assess disease activity if similar findings are observed for these antibodies. In addition, elucidation of the biochemical structure and function of the particular disease-specific antigen at which the immune response is directed may aid in understanding the etiology of the disease and in the development of effective treatments. Also, since these antigens are conserved cellular proteins, they are likely to be functionally important proteins. Study and identification of these antigens may provide significant insights into nuclear and nucleolar processes.
It is therefore an object of this invention to provide DNA encoding antigens that are specifically recognized by myositis-specific autoantibodies which can be expressed in large quantities and the proteins easily purified.
It is a further object of the invention to provide human antigens or portions thereof for use in diagnostic assays and as tools for studying autoimmune myositis.
It is another object of the invention to provide methods for detecting autoantibodies that are uniquely found in the sera of individuals with dermatomyositis or myositis-scleroderma overlap.
Immunoassays for detecting myositis specific antibodies which will help in diagnosis of dermatomyositis (DM), polymyositis (PM), and myositis-sclerosis overlap are provided. Isolated DNA molecules that encode Mi-2 and PM-Scl antigens or antigenic portions thereof, DNA probes for isolating cDNA or genomic DNA clones that encode such antigens or portions thereof, the antigens encoded by the isolated DNA, and diagnostic assays for detecting anti-Mi-2 and anti-PM-Scl autoantibodies in sera are also provided.
DNA that encodes a protein that includes at least one epitope of the human Mi-2 antigen and DNA that encodes the 100-110 kDa subunit of the human PM-Scl antigen have been cloned from a human cDNA library. This DNA can be used to provide proteins that include epitopes of the human Mi-2 and PM-Scl antigens which can be used in assays for autoantibodies to these epitopes, or for other purposes. In addition, this DNA is used to provide probes to screen cDNA and genomic libraries in order to isolate DNA that encodes additional portions of the human antigens or DNA that spans each gene, or to obtain DNA that encodes related antigens in humans and other mammals. The DNA that encodes additional portions of the antigens, and the proteins encoded thereby, may be used in immunodiagnostic assays in order to identify patients that express anti-Mi-2 autoantibodies that do not react with the originally cloned epitopes.
The Mi-2 antigenic epitopes encoded by the DNA, or a portion of them, is useful in immunodiagnostic assays, including ELISAs, dot blots, immunodiffusion, radioimmunoassays and immunoprecipitation assays, to detect anti-Mi-2 to help in diagnosing dermatomyositis. The PM-Scl antigen encoded by the DNA, or a portion of the antigen, is useful in similar assays to help in diagnosing polymyositis and polymyositis-scleroderma overlap disorders.